Methods of treatment using pharmaceutically active ceramide-related compositions

ABSTRACT

This invention provides a compound having the formula R 1  --Y 1  --CHZ 1  --CH(NY 2  Y 3 )--CH 2  --Z 2 , wherein: R 1  is a straight-chained alkyl, alkenyl or alkynyl group having from 8 to 19 carbon atoms in the aliphatic chain; Y 1  is --CH═CH--, --C.tbd.C-- or --CH(OH)CH(OH)--; Z 1  is OH or a conversion-inhibiting group; Z 2  is a conversion-inhibiting group; Y 2  is H, a phenyl group, an alkyl-substituted phenyl group having from 1 to about 6 carbons in the alkyl chain, or an alkyl chain having from 1 to 6 carbons; Y 3  is H or a group having the formula --C(O)R 2  or --S(O) 2  R 2  ; R 2  is a straight-chained alkyl, alkenyl or alkynyl group having from 1 to 23 carbon atoms in the chain; and when Z 2  is an amino, R 2  is an aliphatic chain having from 1 to 9 or from 19 to 23 carbon atoms in the aliphatic chain.

This application is a division of U.S. Ser. No. 08/383,291, now U.S.Pat. No. 5,631,394 filed Feb. 2, 1995, which is a continuation-in-partof U.S. Ser. No. 08/190,295, filed Feb. 2, 1994 now abandoned.

FIELD OF THE INVENTION

This invention is directed to pharmaceutically active sphingolipidcompounds, to liposomes containing pharmaceutically active sphingolipidcompounds, and to methods of using such compounds and liposomes,particularly for the treatment of animals afflicted with cancers.

Cell death in multicellular organisms can be an accidental response toexternal trauma, or it can be a programmed response to internal orexternal stimuli. Necrosis, or accidental cell death, is most often seenwhen cells die uncontrollably as a result of sudden and severe injury toan organism, e.g., by physical or chemical trauma, sustainedhyperthermia or ischemia (see, e.g., J. Cohen, Immunol. Today. 14(3):126(1993)); J. Marx, Science 259:750 (1993)). Plasma membrane damage cancause cells to lose their ability to regulate their osmotic pressure,and cell rupture can thereby result. The consequent leakage of cellcontents can cause further injury to surrounding cells and can invoke aninflammatory response to clear away the cellular debris.

Apoptosis, by contrast, describes a programmed series of eventsresulting in cell death by fragmentation into membrane-bound particles;these particles are then phagocytosed by other cells (see, e.g.,Stedman's Medical Dictionary (Illustrated), supra). Cells typicallyundergo apoptosis in physiologically determined circumstances such asthe elimination of self-reactive T cells, the death of cells (e.g.,neutrophils) with short half-lives, involution of growth factor-deprivedcells, morphogenetic cell death during embryonic development and thedeaths of cellular targets of cell-mediated cytotoxicity (see, e.g., J.Cohen, supra).

Cells undergoing apoptosis can break up into apoptotic bodies, which arecellular fragments that retain their membranes and are able to regulatetheir osmotic pressures. Unlike necrotic cells, there is usually noleakage of cellular contents and hence, no invocation of an inflammatoryresponse. Apoptotic cells typically have disrupted plasma membranes andcondensed, disrupted nuclei. Nuclear chromatin in these cells isfragmented randomly between nucleosomes, as the result of endonucleaseactivation during apoptosis.

Although transcription in apoptotic cells ceases, cell death occurs morerapidly than would be expected from the cessation of transcriptionalone. This indicates that cellular processes in addition totranscription termination are likely to be involved in apoptosis. Geneexpression itself may actually be required for the occurrence of themorphological changes associated with apoptosis (see, e.g., J. Cohen,supra). Alternatively, inhibition of transcription termination mayitself induce apoptosis. Furthermore, apoptosis of some cells does notappear to be affected one way or the other by the inhibition of proteinsynthesis. Expression of the bcl-2 oncogene, for example, can inhibitthe apoptosis otherwise induced by different stimuli, and may therebycontribute to cancer development. Accordingly, inhibition of bcl-2expression may be required to induce apoptosis (see, e.g., J. Marx,supra; J. Cohen, supra; G. Williams and C. Smith, Cell 74:777 (1993); M.Barinaga, Science 259:762 (Feb. 5, 1993)). C-myc protein is known tostimulate cell proliferation; however, it may also stimulate apoptosisin the absence of additional proliferative stimuli. p53, which isthought to suppress tumor growth, may also stimulate apoptosis. C-fas, atransmembrane protein homologous to Tumor Necrosis Factor (TNF), canalso induce apoptosis, as can TNF itself.

TNF is a monokine protein produced by monocytes and macrophages. Thereare two known structurally and functionally related TNF proteins, TNF-αand TNF-β, both of which bind to the same cell surface receptors.Binding to these receptors by TNF leads to the activation of multiplesignal transduction pathways, including the activation ofsphingomyelinase (see, e.g., M. Raines et al., J. Biol. Chem.268(20):14572 (1993); L. Obeid et al., Science 259:1769 (Mar. 12, 1993);H. Morishige et al., Biochim. Biophys. Acta. 1151:59 (1993); J. Vilcekand T. Lee, J. Biol. Chem. 266(12):7313 (1991); Dbaibo et al., J. Biol.Chem. 268(24):17762 (1993); R. Kolesnik, Trends Cell. Biol. 2:232(1992); J. Fishbein et al., J. Biol. Chem. 268(13):9255 (1993)).

Applicants have found that increases in ceramide concentrations canstimulate apoptosis. Ceramides are a class of sphingolipids comprisingfatty acid derivatives of a sphingoid, e.g., sphingosine, base (see,e.g., Stedman's Medical Dictionary (Illustrated), 24th edition (J. V.Basmajian et al., eds.), Williams and Wilkins, Baltimore (1982), p.99)). Different ceramides are characterized by different fatty acidslinked to the sphingoid base. For example, stearic acid can be attachedto the amide group of sphingosine to give rise to the ceramide CH₃(CH₂)₁₂ CH═CH--CHOH--CH(CH₂ OH)--NH--CO--(CH₂)₁₆ CH₃. Shorter- orlonger-chain fatty acids can also be linked to the sphingoid-base.Applicants have also found that attachment of certain chemical groups tosphongolipids and ceramides so as to form analogs of such compounds caninhibit bioconversion of ceramides to sphingomyelins, and can therebylead to an apoptosis stimulating increase in ceramide concentrations.

Ceramides are found in all eukaryotic cell membranes, and are known toparticipate in a variety of critical cellular processes. Furthermore,certain sphingolipid compounds have been found to play a role inprevention of cellular proliferation (). However, none of thesereferences teach applicants' chemical compounds and liposomes, or theiruse in stimulating cell death.

SUMMARY OF THE INVENTION

Provided herein is a compound having the formula R¹ --Y¹ --CHZ¹ --CH(NY²Y³)--CH₂ --Z², wherein: R¹ is a straight-chained alkyl, alkenyl oralkynyl group having from 8 to 19 carbon atoms in the aliphatic chain;Y¹ is --CH═CH--, --C.tbd.C-- or --CH(OH)CH(OH)--; Z¹ is OH or aconversion-inhibiting group; Z² is a conversion-inhibiting group; Y² isH, a phenyl group, an alkyl-substituted phenyl group having from 1 toabout 6 carbons in the alkyl chain, or an alkyl chain having from 1 to 6carbons; Y³ is H or a group having the formula --C(O)R² or --S(O)₂ R² ;R² is a straight-chained alkyl, alkenyl or alkynyl group having from 1to 23 carbon atoms in the chain; and wherein when Z² is an amino, R² isan aliphatic chain having from 1 to 9 or from 19 to 23 carbon atoms inthe aliphatic chain. Preferably, R¹ is an alkyl group, more preferably,CH₃ (CH₂)₁₂ --, Y¹ is --CH═CH--, Y² is H, Y³ is --C(O)R² and R² is analkyl chain.

Conversion-inhibiting groups can have the formula --X² X³ or --O--X² X³,wherein X² is selected from the group consisting of CH₂ --, C(CH₃)₂ --,Si(PO₄)₂ --, Si(CH₃)₂ --, SiCH₃ PO₄ --, C(O)-- and S(O)₂ -- and whereinX³ is selected from the group consisting of --C(O)H, --CO₂ H, --CH₃,--C(CH₃)₃, --Si(CH₃)₃, --SiCH₃ (C(CH₃)₃)₂, --Si(C(CH₃)₃)₃, --Si(PO₄)₂C(CH₃)₃, a phenyl group, an alkyl-substituted phenyl-group having from 1to 6 carbons in the alkyl chain, an alkyl chain having from 1 to 6carbons, an amino moiety, a chlorine, a flourine, or a group having theformula C(R³ R⁴)OH; each of R³ and R⁴ is independently an alkyl chainhaving from 1 to 6 carbons, a phenyl group or an alkyl-substitutedphenyl group having from 1 to 6 carbons in the alkyl chain. Preferably,the conversion-inhibiting group is --OC(O)CH₃, --OC(O)CH₂ CH₂ CH₃,--OC(O)CH(CH₃)CH₃, or --OSi(CH₃)₂ C(CH₃)₃, more preferably; --OSi(CH₃)₂C(CH₃)₃. Conversion-inhibiing groups can also have the formula --X¹ or--OX¹, wherein X¹ is C(O)H, CO₂ H, CH₃, C(CH₃)₃, Si(CH₃)₃, SiCH₃(C(CH₃)₃)₂, Si(C(CH₃)₃)₃, Si(PO₄)₂ C(CH₃)₃, a phenyl group, analkyl-substituted phenyl group having from 1 to 6 carbons in the alkylchain, an alkyl chain having from 1 to 6 carbons, an amino moiety, aflourine, a chlorine, or a group having the formula C(R³ R⁴)OH, and eachof R³ and R⁴ is independently an alkyl chain having from 1 to 6 carbons.

Preferably, the compound of this invention has the formula CH₃ (CH₂)₁₂--CH═CH--CH₂ Z¹ --CH(NHY³)--CH₂ --Z². Y³ is then a group having theformula --C(O)R², more preferably, --C(O)(CH₂)₄ CH₃ --Z² is preferably--OSi(CH₃)₂ C(CH₃)₃, OSi(PO₄)₂ C(CH₃)₃, --C(O)CH₃ or --OC(O)CH₂ CH₂ CH₃.

Also provided herein is a pharmaceutical composition comprising thecompound of this invention and a pharmaceutically acceptable carrier;the composition can also comprise an additional bioactive agent. Furtherprovided is a method of administering a bioactive compound to an animal,preferably a human, which comprises administering to the animal thiscomposition; the method can comprise administering an additionalbioactive agent to the animal.

The animal can be afflicted with a cancer, wherein the method comprisesadministering an amount of the composition which comprises an anticancereffective amount of the compound. Typically, the anticancer effectiveamount of the compound is at least about 0.1 mg of the compound per kgof body weight of the animal. Generally, the anticancer effective amountis from about 1 mg per kg to about 50 mg per kg. Treatable cancersinclude, without limitation, brain, breast, lung, ovarian, colon,stomach or prostate cancers, and can be sarcomas, carcinomas,neuroblastomas, or gliomas. Drug resistant cancers can also be treated.

Provided herein is a liposome having a bilayer which comprises a lipidand a compound having the formula R¹ --Y¹ --CHZ¹ --CH(NY² Y³)--CH₂ --Z2,wherein: R¹ is a straight-chained alkyl, alkenyl or alkynyl group havingfrom 5 to 19 carbon atoms in the chain; Y¹ is --CH═CH--, --C.tbd.C-- or--CH(OH)CH(OH)--; each of Z¹ and Z² is independently OH or aconversion-inhibiting group; Y² is H, a phenyl group, analkyl-substituted phenyl group having from 1 to 6 carbons in the alkylchain, or an alkyl chain having from 1 to 6 carbons; Y³ is H or a grouphaving the formula --R², --C(O)R² or --S(O)₂ R² ; R² is astraight-chained alkyl, alkenyl or alkynyl group having from 1 to 23carbon atoms; and wherein the bilayer comprises at least about five molepercent of the compound. Y³ is preferably R², which is preferably,--(CH₂)₃ CH₃, --(CH₂)₅ CH₃, --(CH₂)₇ CH₃, or --(CH₂)₉ CH₃, and morepreferably, R² is --(CH₂)₅ CH₃, or --C(O)R², which is preferably--C(O)(CH₂)₄ CH₃. Preferably in the liposome of this invention, at leastone of Z¹ and Z² is a conversion-inhibiting group, such as --OC(O)CH₃,--OC(O)CH₂ CH₂ CH₃, --OC(O)CH(CH₃)CH₃, or --OSi(CH₃)₂ C(CH₃)₃. Morepreferably, the conversion-inhibitng group is --OSi(CH₃)₂ C(CH₃)₃. Mostpreferably, the liposome comprises a compound having the formula CH₃--(CH₂)₁₂ --CH═CH--CH₂ Z¹ --CH(NHY³)--CH₂ Z².

Preferably, the liposomal bilayer comprises at least about 10 molepercent of the compound. The bilayer can comprise vitamin D₃ ; suchbilayers preferably comprise about 1 mole percent of vitamin D₃. Thebilayer can also comprise a headgroup-modified lipid. The liposome cancomprise an additional bioactive agent, and can be dehydrated.

Also provided herein is a pharmaceutical composition comprising theliposome of this invention and a pharmaceutically acceptable carrier.Further provided is a method of administering a compound to an animalwhich comprises administering to the animal the composition. The methodcan be used to treat an animal afflicted with a cancer, wherein a doseof the composition is administered and wherein the dose comprises ananticancer effective amount of the liposome. Typically, the dosecomprises at least about 1 mg of the liposome per kg of body weight ofthe animal. Generally, the dose comprises from about 1 mg per kg toabout 1000 mg per kg.

Provided herein is a liposome having a bilayer which comprises a lipidand a compound having the formula R¹ --Y¹ --CHZ¹ --CH(NY² Y³)--CH₂ --Z²,wherein: R¹ is a straight-chained alkyl, alkenyl or alkynyl group havingfrom 5 to 19 carbon atoms in the chain; Y¹ is --CH═CH--. --C.tbd.C-- or--CH(OH)CH(OH)--; each of Z¹ and Z² is independently OH or aconversion-inhibiting group; Y² is H, a phenyl group, analkyl-substituted phenyl group having from 1 to 6 carboins or an alkylchain having from 1 to 6 carbons; Y³ is H or a group having the formula--R², --C(O)R² or --S(O)₂ R² ; R² is a straight-chained alkyl, alkenylor alkynyl group having from 1 to 23 carbon atoms; and wherein thebilayer comprises an anticancer-effective amount of the compound. Alsoprovided is a pharmaceutical composition comprising this liposome and apharmaceutically acceptable carrier. Further provided is a method oftreating an animal afflicted with a cancer which comprises administeringto the animal this composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Ceramide Metabolism. Cer: ceramides; SM:

FIGS. 2A-2D Ceramides Comprising Conversion-inhibiting Group. A: TypeIII Cer-1-TBDMS; C2 Cer-1-TBDMS; C6 Cer-1-TBDMS; C2 Cer-1-TBDPS. B:3-TBDMS C6-Cer; 1-TBDMS C6-Cer; 1,3 DITBDMS C6 Cer (YWI51.a); 1-TBDMS,3-acetate C6-Cer; 1-TBDMS,3-butyrate, C6-Cer. C: 1-acetate-3-one C6-Cer;4,5-diol C6-Cer. D: N-C4 sphingosine; n-hexyl sphingosine; n-C8sphingosine; N-C10 sphingosine.

FIG. 3 Effect of Various Liposomal Ceramide/Sphingomyelin Formulationson the Growth of HL-60 Cells. The number of viable cells (per ml×10,000,y-axis) was determined for lipid doses of 100 μM and 200 μM (z-axis).X-axis: control, egg phosphatidylcholine/cholesterol (EPC/Chol),EPC/Chol/C2-ceramide (C2), EPC/Chol/vitamin D3 (D3), EPC/Chol/D3/C2,EPC/Chol/C6-ceramide (C6), EPC/Chol/D3/C6, SM/Chol and SM/Chol/D3liposomes.

FIG. 4 Effect of Various Liposomal Ceramide/Sphingomyelin Formulationson the Growth of P388 Cells. The number of viable cells (per ml×10,000,y-axis) was determined for lipid doses of 50 μM, 100 μM and 200 μM(z-axis). X-axis: control, egg phosphatidylcholine/ cholesterol(EPC/Chol), EPC/Chol/C2-ceramide (C2), EPC/Chol/vitamin D3 (D3),EPC/Chol/D3/C2, EPC/Chol/C6-ceramide (C6), EPC/Chol/D3/C6, Sphingomyelin(SM)/Chol and SM/Chol/D3 liposomes.

FIG. 5 Effect of Various Liposomal Ceramide/Sphingomyelin Formulationson the Growth of U937 Cells. The number of viable cells (per ml×10,000,y-axis) was determined for lipid doses of 50 μM, 100 μM and 200 μM(z-axis). X-axis: control, egg phosphatidylcholine/ cholesterol(EPC/Chol), EPC/Chol/C2-ceramide (C2), EPC/Chol/vitamin D3 (D3),EPC/Chol/D3/C2, EPC/Chol/C6-ceramide (C6), EPC/Chol/D3/C6, Sphingomyelin(SM)/Chol and SM/Chol/D3 liposomes.

FIG. 6 Effect of Various Liposomal Ceramide/Sphingomyelin Formulationson the Growth of RPMI-7666 Cells. The number of viable cells (perml×10,000, y-axis) was determined for a lipid dose of 200 μM. X-axis:control, egg phosphatidylcholine/ cholesterol (EPC/Chol),EPC/Chol/C2-ceramide (C2), EPC/Chol/vitamin D3 (D3), EPC/Chol/D/C2,EPC/Chol/C6-ceramide (C6), EPC/Chol/D3/C6, Sphingomyelin (SM)/Chol andSM/Chol/D3 liposomes.

FIG. 7 Effect of Liposomal Ceramide on the Growth of Normal (RPMI-7666)and Cancer (U-937) Cells. The number of viable cells in each of thecultures was determined and is given as the percent relative to thecontrol (y-axis).

FIG. 8 Therapeutic Efficacy of Liposomal Ceramides in Mice. X-axis: dayspost-liposome/control administration; y-axis: percent survival intreatment group. Circle-in-square: control mice administered HEPESbuffered saline; filled square liposomal vitamin D3; open diamond:liposomal C2 ceramide; filled diamond: liposomal C6 ceramide.

FIGS. 9A-9E In Vitro Sensitivity of A549 Cells to C6-CeramideDerivatives. A: one-hour exposure; B: four-hour exposure; C: eight-hourexposure; D: twenty four-jour exposure; E: forty eight-hour exposureX-axis: drug (ceramide derivative) concentration (micromolar); y-axis:percent cell growth. "X": N-C8 sphingosine; diamond: 1-acetate,C6-ceramide; square: 1-butyrate, C6-ceramide; triangle: 1-isobutyrate,C6-ceramide.

FIG. 10 In Vitro Sensitivity of A54.9 Cells to 1-Acetate, C6-Ceramidefor Different Lengths of Exposure. X-axis: drug concentration(micromolar); y-axis: percent growth. Diamond: one hour; square: fourhours; triangle: eight hours; X: twenty four hours; star: forty eighthours.

FIG. 11 In Vitro Sensitivity of A549 Cells to 1-Butyrate, C6-Ceramidefor Different Lengths of Exposure. X-axis: drug concentration(micromolar); y-axis: percent growth. Diamond: one hour; square: fourhours; triangle: eight hours; X: twenty four hours; star: forty eighthours.

FIG. 12 In Vitro Sensitivity of A549 Cells to 1-Isobutyrate, C6-Ceramidefor Different Lengths of Exposure. X-axis: drug concentration(micromolar); y-axis: percent growth. Diamond: one hour; square: fourhours; triangle: eight hours; X: twenty four hours; star: forty eighthours.

FIG. 13 In Vitro Sensitivity of A549 Cells to N-C8 Sphingosine forDifferent Lengths of Exposure. X-axis: drug concentration (micromolar);y-axis: percent growth. Diamond: one hour; square: four hours; triangle:eight hours; X: twenty four hours; star:. forty eight hours.

FIG. 14 Therapeutic Efficacy of N-hexyl Sphingosine Against P-388/ADR(adriamycin-resistant) Tumor-Bearing Mice. X-axis: days after therapy;y-axis: percent survival in treatment group. "+": Control (untreated);squares: one dose of 20 mg n-hexyl sphingosine per kg of body weight;circles: 3 20 mg/kg doses.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is a compound having the formula R¹ --Y¹ --CHZ1--CH(NY²Y³)CH₂ --Z², wherein: R¹ is a straight-chained alkyl, alkenyl or alkynylgroup having from 8 to 19 carbon atoms in the aliphatic chain; Y¹ is--CH═CH--, --C.tbd.C-- or --CH(OH)CH(OH)--; Z¹ is OH or aconversion-inhibiting group; Z² is a conversion-inhibiting group; Y² isH, a phenyl group, an alkyl-substituted phenyl group having from 1 toabout 6 carbons in the alkyl chain, or an alkyl chain having from 1 to 6carbons; Y³ is H or a group having the formula --C(O)R₂ or --S(O)₂ R² ;R² is a straight-chained alkyl, alkenyl or alkynyl group having from 1to 23 carbon atoms in the chain; and wherein when Z² is an amino, R² isan aliphatic chain having from 1 to 9 or from 19 to 23 carbon atoms inthe aliphatic chain. Preferably, R¹ is an alkyl group, more preferably,CH₃ (CH₂)12--, Y¹ is --CH═CH--, Y² is H, Y³ is --C(O)R² and R² is analkyl chain, more preferably an alkyl chain having from 6 to 8 carbons.Most preferably, R¹ and R² together comprise from about 15 to about 25carbons, wherein R¹ preferably comprises 13 carbons and R² preferablycomprises 6 to 8 carbons. Without intending to be limited by theory, itis believed that total carbon chain length of a lipid is an importantfactor in determing the ability of the lipid to insert itself intobiological membranes.

Without intending for this invention in any way to be limited by theory,it is believed that sphingosines and ceramides can act as signaltransducers or secondary messengers in cells, i.e., that intracellularlevels are increased in response to external stimuli, and that thisincrease results in enhanced protein kinase and phosphatase activities(see. e.g., M. Raines et al., supra; R. Kolesnik et al., supra; G.Dbaibo et al., supra; and J. Fishbein et al., supra). Activated proteinkinases and phosphatases can activate cellular processes which lead tocell death. Accordingly, it can be therapeutically desirable to increaseintracellular concentrations of sphingosines and ceramides in cancercells.

Sphingosines and ceramides are formed in animal cells by the combinationof palmitoyl CoA (CH₃ (CH₂)₁₄ --CO--S--CoA) and serine to givedehydrosphinganine (CH₃ (CH₂)₁₄ Co--CH(NH₃)--CH₂ OH and CO₂ (see, e.g.,L. Stryer, Biochemistry (2nd edition), W. H. Freeman and Co., New York,pp. 461-462)). Dehydrosphinganine is converted to dihydrosphingosine(CH₃ (CH₂)₁₄ --CH(OH)--CH(NH₃)--CH₂ OH) which is then converted tosphingosine (CH₃ (CH₂)₁₂ CH═CH--CH(OH)--CH(NH3)--CH₂ OH). A fatty acidis then linked to the amide group of sphingosine to give rise to aceramide (CH₃ (CH₂)₁₂ CH═CH--CHOH--CH(CH₂ OH)--NH--CO--R, where R is afatty acid chain). A phosphorylcholine group (PO₄ CH₂ CH₂ --N(CH₃)₃) canbe attached to the ceramide at its hydroxyl group to produce asphingomyelin (CH₃ (CH₂)₁₂ CH═CH--CHOH--CH(CH₂ PO₄ CH₂ CH₂--N(CH₃)₃)--NH--CO--R). Sphingomyelinase can catalyze the hydrolyticremoval of the phosphorylcholine from the sphingomyelin to give rise toa ceramide (see, e.g., FIG. 1). Reverse hydrolysis of the ceramide cangive rise to a sphingomyelin.

Blockage or inhibition of this "reverse hydrolysis" step, that is,conversion of a ceramide to the corresponding sphingomylein, can lead toincreased intracellular ceramide levels. "Conversion-inhibiting groups"are attached to sphingosines and ceramides to inhibit sphingomyleinformation therefrom. Such groups are generally not found atached tosphingosines and ceramides, or their biosynthetic precursors, in animalcells

The compounds of this invention are synthesized by a number of routeswell known to and readily practiced by ordinarily skilled artisans,given the teachings of this invention (see, for example, below, wherein"rf" refers to one of the following references: 1: J. Am. Chem. Soc.,94:6190 (1972); 2: J. Org. Chem. 59:668 (1994); 3: Angew. Chem., Intl.Ed. (English), 17:569 (1978); 4: Vogel's Textbook of Practical OrganicChemistry (5th ed.), pp. 769-780); 5: J. Org. Chem. 40:574 (2975); 6: J.Org. Chem. 59:182 (1994); 7: J. Org. Chem. 25:2098 (1960); 8: Synthesis(1985): pp. 253-268; 9: J. Chem. Soc. (1953): p. 2548; 10: J. Am. Chem.Soc. 90:4462, 4464 (1968); 11: Oxidations in Organic Chemistry (Am.Chem. Soc, Washington, D.C. (1990), pp. 60-64; 12: J. Med. Chem. 30 1326(1987); 13: Synth. Commun. 9:757 (1979); 14: The Chemistry of Amides (J.Wiley & Sons, New York (2970)), pp. 795-801; 15: J. Med. Chem. 37:2896(1994);4: J. Med. Chem. 30:1326 (1987); 16: Rec. Chem. Prog. 29:85(1968); and 17: Phospholipids Handbook (Marcell Dekker, Inc., New York(1993), p. 97); the contents of these are incorporated herein byreference).

For example, such artisans would use a sphingosine or a ceramide astheir starting material. Alkyl, alkenyl or alkynyl chains of varyinglength can be attached thereto, or removed therefrom, by known means.Conversion-inhibiting groups can also be attached to the sphingosinesand ceramides by known means. These include, without limitation,oxidation/reduction, substitution, condensation and alkylationreactions, as well as other generally accepted means for attaching andremoving chemical groups from a compounds and for converting compoundsfrom one form to another. Such reactions are generally formed usinggenerally accepted solvents and are performed under readily determinableconditions. ##STR1##

Specific compounds can be synthesized as follows. Synthesis of silyether of ceramide: a mixture of ceramide and t-butyldimethylsilylchloride (1 equivalent) and imidazole (2 equivalent) in DMF is stirredunder N₂ at room temperature overnight. The solvent is then removedunder a stream of N₂ and residue is dissolved in CH₂ Cl₂, washed (H₂ O),dried (MgSO₄) and concentrated to dryness. The residue is purified oversilica gel (AcOEt: Hexan=1:3). Synthesis of 1-ester ceramide: Themixture of ceramide and Ac2O (1 equivalent) and catalytic amount ofdimethyl amino pyridine in dry CH2Cl2 is stirred at room temperature for1 hour and the reaction is checked by TLC (AcOEt). The mixture is thenconcentrated. The crude product is purified over silica gel (AcOEt:Hexane=2:3.5) Oxidation of C3-OH of ceramide to ketone: 1-OAc ceramideis dissolved in acetone and cooled in an ice-bath. Jone's reagent isdropwised slowly till the orange color persists. The reaction isquenched by isopropanol, and NaHCO₃ is added and stirred for 5 minutes.The solution is filtered and concentrated to dryness. The crude productwas purified by preparative TLC (ACOET: Hexane=1:2.5). Reduction ofceramide to sphingosine analogs: To an ice-cold stirred solution ofceramide in anhydrous THF is added LiAIH₄ and the mixture is stirred atroom temperature under N₂ for 24 hours. Under ice cooling, the reactionmixture is quenched by addition of saturated aqueous NaHCO₃. Theresulting slurry is filtered and washed with THF. The solution isconcentrated and the residue is brought into CH₂ Cl₂, washed with H₂ O,dried (MgSO₄) and concentrated to dryness. The residue is then purifiedover preparative TLC (silica gel) CH₂ Cl₂ : MeOH: TEA=8:1 0.08.Synthesis of 4,5-diol ceramide: To a solution of ceramide in a mixtureof Me₂ CO distilled H₂ O and t-BuOH, N-Methyl morpholine N-oxide (NMO,1.2 equivalent) and OsO₄ (catalytic amount) in THF are added. Thereaction mixture is stirred at 45° C. for 6 hours, quenched by solidNaHCO₃, and the mixture is then stirred for 15 minutes. The suspensionis filtered, and the filtrate dissolved in THF; the solution is thenwashed with brine. The organic solution is separated, dried andconcentrated to dryness. The residue is purified over preparative TLC(THF).

Suitable conversion-inhibiting groups can be identified by a number ofmeanns readily practiced by ordinarily skilled artisans, given themotivation by this invention to identify such groups. For example, andwithout limitation, such artisans can select a chemical moiety, andattach it to a sphingosine or ceramide as described above. The artisanscan then readily determine the relative rate at which such a compoundundergoes hydrolysis, and the rate at which a sphingmyelin is formedfrom the compound. Rates of hydrolysis are themselves readilydeterminable by ordinarily skilled artisans, for example and withoutlimitation, by attaching a radioactive moiety to a sphingosine orceramide and then following the rate of hydrolytic cleavage of themoiety by chromatographic means. Rates of sphingomyelin formation arealso readily determinable, for example and without limitation, bycombining radioactive phosphorylcholine with a conversion-inhibitinggroup-containing compound in an enzyme system capable of attaching thephosphorylcholine to the compound, and then using chromatographic meansto assess the rate at which the phosphorylcholine is added. PreferredConversion-inhibiting groups are those which most inhibit hydrolysis andphosphorylcholine attachment.

Conversion-inhibiting groups can have the formula --X² X³ or --O--X² X³,wherein X² is selected from the group consisting of CH₂ --, C(CH₃)₂ --,Si(PO₄)₂ --, Si(CH₃)₂ --, SiCH₃ PO₄ --, C(O)-- and S(O)₂ --and whereinX³ is selected from the group consisting of --C(O)H, --CO₂ H, --CH₃,--C(CH₃)₃, --Si(CH₃)₃, --SiCH₃ (C(CH₃)₃)₂, --Si(C(CH₃)₃)₃, --Si(PO₄)₂C(CH₃)₃ a phenyl group, an alkyl-substituted phenyl group having from 1to 6 carbons in the alkyl chain, an alkyl chain having from 1 to 6carbons, an amino moiety, a chlorine, a flourine, or a group having theformula C(R³ R⁴)OH; each of R³ and R⁴ is independently an alkyl chainhaving fron 1 to 6 carbons, a phenyl group or an alkyl-substitutedphenyl group having from 1 to 6 carbons in the alkyl chain. Preferably,the conversion-inhibiting group is --OC(O)CH₃, --OC(O)CH₂ CH₂ CH₃,--OC(O)CH(CH₃)CH₃, or --OSi(CH₃)₂ C(CH₃)₃ (TBDMS) more preferably,--OSi(CH₃)₂ C(CH₃)₃.

Conversion-inhibiting groups can also have the formula --X¹ or --OX¹,wherein X¹ is C(O)H, CO₂ H, CH₃, C(CH₃)₃, Si(CH₃)₃, SiCH₃ (C(CH₃)₃)₂,Si(C(CH₃)₃)₃, Si(PO₄)₂ C(CH₃)₃, a phenyl group, an alkyl-substitutedphenyl group having from 1 to 6 carbons in the alkyl chain, an alkylchain having from 1 to 6 carbons, an amino moiety, a flourine, achlorine, or a group having the formula C(R³ R⁴)OH; each of R³ and R⁴ isindependently an alkyl chain having from 1 to 6 carbons.Conversion-inhibiting groups include attachment of chemical moities tosphingosines and ceramides by ether, silyl ether, ester, acetal andsulfonate linkages.

Preferably, the compound of this invention has the formula CH₃ (CH₂)₁₂--CH═CH--CH₂ Z¹ --CH(NHY³),CH₂ --Z². Y³ is then prefeably a group havingthe formula --C(O)R², more preferably, --C(O)(CH₂)₄ CH₃. Z² ispreferably --OSi(CH₃)₂ C(CH₃)₃, --OSi(PO₄)₂ C(CH₃)₃, --C(O)CH₃ or--OC(O)CH₂ CH₂ CH₃.

Also provided herein is a pharmaceutical composition comprising thecompound of this invention and a pharmaceutically acceptable carrier;the composition can also-comprise an additional bioactive agent."Pharmaceutically acceptable carriers" as used herein are generallyintended for use in connection with the administration of lipids andliposomes, including liposomal bioactive agent formulations, to animals,including humans. Pharmaceutically acceptable carriers are generallyformulated according to a number of factors well within the purview ofthe ordinarily skilled artisan to determine and account for, includingwithout limitation: the particular liposomal bioactive agent used, itsconcentration, stability and intended bioavailability; the disease,disorder or condition being treated with the liposomal composition; thesubject, its age, size and general condition; and the composition'sintended route of administration, e.g., nasal, oral, ophthalmic,topical, transdermal, vaginal, subcutaneous, intramammary,intraperitoneal, intravenous, or intramuscular (see, for example, Nairn(1985)). Typical pharmaceutically acceptable carriers used in parenteralbioactive agent administration include, for example, D5W, an aqueoussolution containing. 5% weight by volume of dextrose, and physiologicalsaline. Pharmaceutically acceptable carriers can contain additionalingredients, for example those which enhance the stability of the activeingredients included, such as preservatives and anti-oxidants.

Further provided is a method of administering a bioactive compound to ananimal, preferably a human, which comprises administering to the animalthis composition; the method can comprise administering an additionalbioactive agent to the animal. The administration, which can be by anymeans generally accepted for administering pharmaceutical products toanimals, is generally intravenous administration.

The animal can be afflicted with a cancer, wherein the method comprisesadministering an amount of the composition which comprises an anticancereffective amount of the compound. Treatable cancers include, withoutlimitation, brain, breast, lung, ovarian, colon, stomach or prostatecancers, and can be sarcomas, carcinomas, neuroblastomas, or gliomas,amongst others. Drug resistant cancers can also be treated.

"Anticancer effective amounts" of the compound of this invention aregenerally amounts effective to inhibit, ameliorate, lessen or preventestablishment, growth, metastasis or invasion of one or more cancers inanimals to which the compound has been administered. Anticancereffective amounts are generally chosen in accordance with a number offactors, e.g., the age, size and general condition of the subject, thecancer being treated and the intended route of administration, anddetermined by a variety of means, for example, dose ranging trials, wellknown to, and readily practiced by, ordinarily skilled artisans giventhe teachings of this invention. Typically, the anticancer effectiveamount of the compound is at least about 0.1 mg of the compound per kgof body weight of the animal. Generally, the anticancer effective amountis from about 1 mg per kg to about 50 mg per kg.

Provided herein is a liposome having a bilayer which comprises a lipidand a compound having the formula R¹ --Y¹ --CHZ¹ --CH(NY² Y³)--CH₂ --Z²,wherein: R¹ is a straight-chained alkyl, alkenyl or alkynyl group havingfrom 5 to 19 carbon atoms in the chain; Y¹ is --CH═CH--, --C.tbd.C-- or--CH(OH)CH(OH)--; each of Z¹ and Z² is independently OH or aconversion-inhibiting group; Y² is H, a phenyl group, analkyl-substituted phenyl group having from 1 to 6 carbons in the alkylchain, or an alkyl chain having from 1 to 6 carbons; Y³ is H or a grouphaving the formula --R², --C(O)R² or --S(O)₂ R² ; R² is astraight-chained alkyl, alkenyl or alkynyl group having from 1 to 23carbon atoms; and wherein the bilayer comprises at least about five molepercent of the compound.

Liposomes are self-assembling structures comprising one or more lipidbilayers, each of which surrounds an aqueous compartment and comprisestwo opposing monolayers of amphipathic lipid molecules. These comprise apolar (hydrophilic) headgroup region covalently linked to one or twonon-polar (hydrophobic) acyl chains. Energetically unfavorable contactsbetween the hydrophobic acyl chains and the aqueous medium are generallybelieved to induce lipid molecules to rearrange such that the polarheadgroups are oriented towards the aqueous medium while the acyl chainsreorient towards the interior of the bilayer. An energetically stablestructure is formed in which the acyl chains are effectively shieldedfrom coming into contact with the aqueous medium.

Liposomes can be made by a variety of methods (for a review, see, forexample, Deamer and Uster (1983)). These methods include withoutlimitation: Bangham's methods for making muiltilamellar liposomes(MLVs); Lenk's, Fountain's and Cullis' methods for making MLVs withsubstantially equal interlamellar solute distribution (see, for example,U.S. Pat. Nos. 4,522,803, 4,588,578, 5,030,453, 5,169,637 and4,975,282); and Paphadjopoulos et al.'s reverse-phase evaporation method(U.S. Pat. No. 4,235,871) for preparing oligolamellar liposomes.Unilamellar vesicles can be produced from MLVs by such methods assonication (see Paphadjopoulos et al. (1968)) or extrusion (U.S. Pat.No. 5,008,050 and U.S. Pat. No. 5,059,421). The ether lipid liposome ofthis invention can be produced by the methods of any of thesedisclosures, the contents of which are incorporated herein by reference.

Various methodologies, such as sonication, homogenization, French Pressapplication, milling and extrusion can be used to size reduce liposomes,that is to prepare liposomes of a smaller size from larger liposomes.Tangential flow filtration (see WO89/008846), can also be used toregularize the size of liposomes, that is, to produce liposomes having apopulation of liposomes having less size heterogeneity, and a morehomogeneous, defined size distribution. The liposome of this inventioncan be unilamellar or multilamellar.

Liposomal bilayers can comprise a variety of ampipathic lipids,including those which are saturated or unsaturated, and which typicallyhave acyl chains of from 10 to 24 carbons. Suitable polar groupsinclude, without limitation, phosphorylcholine, phosphorylethanolamine,phosphorylserine, phosphorylglycerol and phosphorylinositiol. Suitableacyl chains include, without limitation, laurate, myristate, palmitate,stearate and oleate chains. Liposomal bilayers can further comprisesterols, such as cholesterol. Sterols generally affect the fluidity oflipid bilayers, typically increasing the fluidity, of bilayerhydrocarbon chains below the gel-to-liquid transition temperature (Tm),and decreasing fluidity above the Tm (see, for example, Lewis andMcElhaney (1992) and Darnell et al. (1986)) Accordingly, sterolinteractions with surrounding hydrocarbon chains generally inhibitemigration of these chains from the bilayer.

Preferably, the liposomal bilayer comprises at least about 10 molepercent of the compound. When Y³ is R², it is then preferably, --(CH₂)₃CH₃, --(CH₂)₅ CH₃, --(CH₂)₇ CH₃, or --(CH₂)₉ CH₃, and more preferably,(CH₂)₅ CH₃. When Y³ is --C(O)R², it is then preferably --C(O)(CH₂)₄ CH₃.Preferably, the liposome comprises a compound having the formula CH₃--(CH₂)₁₂ --CH=CH--CH₂ Z¹ --CH(NHY³)--CH₂ Z² ; more preferably, thecompound comprises at least one conversion-inhibiting group, such as--OC(O)CH₃, --OC(O)CH₂ CH₂ CH₃, --OC(O)CH(CH₃)CH₃, or --OSi(CH₃)₂C(CH₃)₃. More preferably, the conversion-inhibitng group is --OSi(CH₃)₂C(CH₃)₃ (TBDMS).

Intracellular ceramide levels can also be increased by administration ofvitamin D3, either separately from administration of the compounds andliposomes of this invention, or more preferably, in connection with theadministration of liposomes. Without intending in any way to be limitedby theory, it is believed that vitamin D3 can stimulate sphingomyelinaseto convert sphingomyelins to ceramides. Preferably, bilayers containingvitamin D3 contain about 1 mole percent of vitamin D₃.

The liposome can comprise an additional bioactive agent. A "bioactiveagent," is any compound or composition of matter that can beadministered to animals, preferably humans. Such agents can havebiological activity in animals; the agents can also be useddiagnostically in the animals. Bioactive agents include therapeutic andimaging agents. Bioactive agents which may be associated with liposomesinclude, but are not limited to: antiviral agents such as acyclovir,zidovudine and the interferons; antibacterial agents such asaminoglycosides, cephalosporins and tetracyclines; antifungal agentssuch as polyene antibiotics, imidazoles and triazoles; antimetabolicagents such as folic acid, and purine and pyrimidine analogs;antineoplastic agents such as the anthracycline antibiotics and plantalkaloids; sterols such as cholesterol; carbohydrates, e.g., sugars andstarches; amino acids, peptides, proteins such as cell receptorproteins, immunoglobulins, enzymes, hormones, neurotransmitters andglycoproteins; dyes; radiolabels such as radioisotopes andradioisotope-labelled compounds; radiopaque compounds; fluorescentcompounds; mydriatic compounds; bronchodilators; local anesthetics; andthe like. Liposomal bioactive agent formulations can enhance thetherapeutic index of the bioactive agent, for example by buffering theagent's toxicity. Liposomes can also reduce the rate at which abioactive agent is cleared from the circulation of animals. Accordingly,liposomal formulation of bioactive agents can mean that less of theagent need be administered to achieve the desired effect. Additionalbioactive agents preferred for the liposome of this invention includeantimicrobial, anti-inflammatory and antineoplastic agents ortherapeutic lipids, for example, ceramides. Most preferably, theadditional bioactive agent is an antineoplastic agent.

Liposomes can be loaded with one or more biologically active agents bysolubilizing the agent in the lipid or aqueous phase used to prepare theliposomes. Alternatively, ionizable bioactive agents can be loaded intoliposomes by first forming the liposomes, establishing anelectrochemical potential, e.g., by way of a pH gradient, across theoutermost liposomal bilayer, and then adding the ionizable agent to theaqueous medium external to the liposome (see Bally et al. U.S. Pat. No.5,077,056 and WO86/01102).

The liposome of this invention can comprise a headgroup-modified lipid."Headgroup-modified lipids" are lipids which, when incorporated into thelipid bilayers of liposomes can inhibit clearance of the liposomes fromthe circulatory systems of animals to which they have been administered.Liposomes are cleared from an animal's body by way of itsreticuloendothelial system (RES) which consists of fixed and circulatingmacrophages. Avoiding RES clearance can allow liposomes to remain in thecirculation longer, meaning that less of the drug need be administeredto achieve desired serum levels. Enhanced circulation times can alsoallow targeting of liposomes to non-RES containing tissues. Liposomalsurfaces can become coated with serum proteins when administered toanimals, i.e., liposomes can be opsonized. Rates of clearance by the REScan be related to the rate and level of opsonization; accordingly,clearance can be inhibited by modifying the outer surface of liposomessuch that binding of serum proteins is generally inhibited. This can beaccomplished by minimizing or shielding negative surface charges, whichcan promote protein binding, or by otherwise presenting a sterichindrance to the binding of serum proteins.

Effective surface modification, that is, alterations to the outersurfaces of liposomes which result in inhibition of opsonization and RESuptake, can be accomplished by incorporating headgroup-modified lipidsinto liposomal bilayers. "Headgroup-modified lipids" as used herein areamphipathic lipids whose polar headgroups have been derivatized byattachment thereto of a chemical moiety, e.g., polyethylene glycol, apolyalkyl ether, a ganglioside, an organic dicarboxylic acid or thelike, which can inhibit the binding of serum proteins to liposomes suchthat the pharmacokinetic behavior of the vesicles in the circulatorysystems of animals is altered (see, e.g., Blume et al., Biochim.Biophys. Acta. 1149:180 (1993); Gabizon et al., Pharm. Res. 10(5):703(1993); Park et al. Biochim. Biophys Acta. 1108:257 (1992); Woodle etal., U.S. Pat. No. 5,013,556; Allen et al., U.S. Pat. Nos. 4,837,028 and4,920,016; U.S. Ser. No. 142,691, filed Oct. 25, 1993; the contents ofthese disclosures are incorporated herein by reference).

The amount of a headgroup-modified lipid incorporated into the liposomedepends upon a number of factors well known to the ordinarily skilledartisan, or within his purview to determine without undueexperimentation. These include, but are not limited to: the type oflipid and the type of headgroup modification; the type and size of theliposome; and the intended therapeutic use of the liposomal formulation.Typically, the concentration of the headgroup-modified lipid in thelipid bilayer of the liposome is at least about five mole percent,desirably, about ten mole percent.

The liposome of this invention can be dehydrated, stored and thenreconstituted such that a substantial portion of their internal contentsare retained in the liposomes. Liposomal dehydration generally requiresuse of a hydrophilic drying protectant (see U.S. Pat. Nos. 4,229,360 and4,880,635). This hydrophilic compound is generally believed to preventthe rearrangement of the lipids in the liposome, so that the size andcontents are maintained during the drying procedure and throughrehydration, such that the liposomes can be reconstituted. Appropriatequalities for such drying protectants are that they be strong hydrogenbond acceptors, and possess stereochemical features that preserve theintramolecular spacing of the liposome bilayer components. Saccharidesugars, preferentially mono- and disaccharides, are suitable dryingprotectants for liposomes. Alternatively, the drying protectant can beomitted if the liposome preparation is not frozen prior to dehydration,and sufficient water remains in the preparation subsequent todehydration.

Also provided herein is a pharmaceutical composition comprising theliposome of this invention and a pharmaceutically acceptable carrier.Further provided is a method of administering a compound to an animalwhich comprised administering to the animal thiscomposition. The methodcan be used to treat an animal afflicted with a cancer, wherein a doseof the composition is administered and wherein the dose comprises ananticancer effective amount of the liposome. Typically, the dosecomprises at least about 1 mg of the liposome per kg of body weight ofthe animal. Generally, the dose comprises from about 1 mg per kg toabout 1000 mg per kg.

Provided herein is a liposome having a bilayer which comprises a lipidand a compound having the formula R¹ --Y¹ --CHZ¹ --CH(NY² Y³)--CH₂ --Z²,wherein: R¹ is a straight-chained alkyl, alkenyl or alkynyl group havingfrom 5 to 19 carbon atoms in the chain; Y¹ is --CH═CH--, --C.tbd.C-- or--CH(OH)CH(OH)--; each of Z¹ and Z² is independently OH or aconversion-inhibiting group; Y² is H, a phenyl group, analkyl-substituted phenyl group having from 1 to 6 carboins or an alkylchain having from 1 to 6 carbons; Y³ is: H or a group having the formula--R², --C(O)R² or --S(O)₂ R² ; R² is a straight-chained alkyl, alkenylor alkynyl group having from 1 to 23 carbon atoms; and wherein thebilayer comprises an anticancer-effective amount of the compound. Alsoprovided is a pharmaceutical composition comprising this liposome and apharmaceutically acceptable carrier. Further provided is a method oftreating an animal afflicted with a cancer which comprises administeringto the animal this composition.

EXAMPLES Example 1

Liposome Preparation

Liposomes were prepared with the components, and at the mole ratios ofcomponents, indicated in Table 1 (see below) by the solvent evaporationmethod. For example, PC/Chol/C2-ceramide liposomes were prepared bydissolving 1.8242 mg bovine phosphatidylcholine (BPC), 0.4639 mgcholesterol (Chol) and 0.1366 mg C2-ceramide (C2) in achloroform/methanol solvent mixture. (2:1, volume/volume). The solventwas then evaporated to produce dried lipid, and the dried lipid wasrehydrated with HEPES buffered saline (10 mM HEPES, 150 mM NaCl, pH7.4). For vitamin D3-containing preparations, 0.0154 mg vitamin D3 wasadded to the lipid mixture. For C6-ceramide-containing preparations,0.1590 mg C6 ceramide (C6) was substituted for the C2 ceramide. Forsphingomyelin (SM)-containing preparations, 2.0470 mg SM, 0.4639 mgcholesterol and 0.0154 mg vitamin D3 were used. Furthermore, the PC/Choland PC/Chol/D3 preparations were prepared with 2.1280 mg BPC, 0.4639 mgcholesterol and 0.0154 mg vitamin D3.

                  TABLE 1                                                         ______________________________________                                        LIPOSOME PREPARATION                                                          COMPONENTS          MOLAR RATIO                                               ______________________________________                                        PC:Chol:C2          6:3:1                                                     PC:Chol:C2:D3       6:3:1:0.1                                                 PC:Chol:C6          6:3:1                                                     PC:Chol:C6:D3       6:3:1:0.1                                                 SM:Chol             7:3                                                       SM:Chol:D3          7:3:0.1                                                   PC:Chol             7:3                                                       PC:Chol:D3          7:3:1:0.1                                                 ______________________________________                                         PC: phosphatidylcholine; Chol: cholesterol; C2: C2 ceramide; D3: vitamin      D3; C6: C6 ceramide; SM: sphingomyelin.                                  

Example 2

Effect Of Various Liposomal Ceramide/Sphingomyelin Formulations on theGrowth of HL-60 Cells

2×10⁵ HL-60 cells were incubated with eggphosphatidylcholine/cholesterol (EPC/Chol), EPC/Chol/C2-ceramide (C2),EPC/Chol/Vitamin D3 (D3), EPC/Chol/D3/C2, EPC/Chol/C6-ceramide (C6),EPC/Chol/D3/C6, sphingomyelin (SM)/Chol and SM/Chol/D3 liposomes, aswell as with buffer (no liposomes; "control") and with eggphosphatidylcholine/cholesterol (EPC/Chol) liposomes. Incubation was at37 degrees C in serum-free medium, supplemented with 5 mg/L insulin and5 mg/L transferrin, for 24 hours. Fetal calf serum was then added to theculture medium to a final concentration of 10%; the cells were thenincubated for another 24 hours, after which the number of viable cellsin each culture were counted using trypan blue staining and ahemocytometer. The number of viable cells was determined for lipid dosesof 100 μM and 200 μM, and is given, in the figures (below) as the numberof viable cells per ml of culture medium, times 10,000. Results arereported in FIG. 3 and Table 2 (see below).

Example 3

Effect of Various Liposomal Ceramide/Sphingomyelin Formulations on theGrowth of P388 Cells

2×10⁵ P388 cells were incubated with various ceramide or sphingomyelinliposomal formulations (see Example 2, above), as well as with bufferalone and with egg phosphatidylcholine/cholesterol (EPC/Chol) liposomes,under the conditions given above. The number of viable cells in thecultures was determined for lipid doses of 50 μM, 100 μM and 200 μM.Results are reported in FIG. 4 and Tables 2 and 3.

Example 4

Effect of Various Liposomal Ceramide/Sphingomyelin Formulations on theGrowth of U937 Cells

2×10⁵ U937 cells were incubated with the various ceramide orsphingomyelin liposomal formulations indicated above (see Example 2), aswell as with buffer alone and with egg phosphatidyicholine/cholesterol(EPC/Chol) liposomes, under the conditions given above. The number ofviable cells in the cultures was determined for lipid doses of 50 μM;100 μM and 200 μM. Results are reported in FIGS. 5 and 7, and Tables 2and 3.

Example 5

Effect of Various Liposomal Ceramide/Sphingomyelin Formulations on theGrowth of RPMI-7666 Cells

2×10⁵ RPMI-7666 cells were incubated with the variousceramide/sphingomyelin liposomal formulations indicated above (seeexample 2), as well as with no liposomes (control) and with eggphosphatidylcholine/cholesterol (EPC/Chol) liposomes, under theconditions indicated above. The number of viable cells in the cultureswas determined. Results are reported in FIGS. 6 and 7, and Tables 2 and3.

Example 6

Effect of Various Liposomal Ceramide/Sphingomyelin Formulations on theGrowth of CHO/K1 Cells

2×10⁵ CHO/k1 cells were incubated with the variousceramide/sphingomyelin liposomal formulations indicated above (seeexample 2), as well as with no liposomes (control) and with eggphosphatidylcholine/cholesterol (EPC/Chol) liposomes, under theconditions indicated above. The number of viable cells in the cultureswas determined. Results are reported in Table 2.

                  TABLE 2                                                         ______________________________________                                        Survival of Various Cancer Lines (Without Serum)                              % Survival (By trypan blue exclusion assay)                                   Formulations                                                                           RPMI-7666 U-937    P-388  HL-60 CHO/k1                               ______________________________________                                        BPC/CHOL/                                                                              129.7, 107.1                                                                            113.4, 130                                                                             136, 103                                                                             87    138                                  VD3                                                                           Control  100       100      100    100   100                                  BPC/CHOL/                                                                              103, 90   63.5, 55 49.6, 26                                                                             37    73                                   VD 3/C-6                                                                      FREE C-6 79        82.6     55.4   --    --                                   CERAMIDE                                                                      FREE C6 m-                                                                             85.6      80.6     --     --    --                                   silyl-ester                                                                   BPC/CHOL/                                                                              93.7      49       46     47    90                                   VD 3/C-2                                                                      ______________________________________                                         Bioactive lipid dose = 20 uM                                             

                  TABLE 3                                                         ______________________________________                                        Effect of Free and Liposomal ceramide on                                      various cancer lines (With serum)                                             % Survival (By trypan blue assay)                                             Formulations RPMI-7666  U-937     P-388                                       ______________________________________                                        Control      100        100       100                                         BPC/CHOL/VD3/C-6                                                                           93.8       84.3      55.0                                                     82 (thy)*  75.1 (thy)*                                                                             84.7 (thy)*                                 FREE C-6 CERAMIDE                                                                          89.2       97        84.5                                        ______________________________________                                         Bioactive lipid dose = 20 uM;                                                 *growth inhibition measured by standard thymidine incorporation assay.   

Example 7

Therapeutic Efficacy of Liposomal Ceramides in Mice

CDF1 mice were each injected intraperitoneally with 2.5×10⁶ P388 cells.Groups having ten mice each were intraperitoneally administered either aHEPES-buffered saline control (1.0 mM HEPES, 150 mM NaCl, pH 7.4), orliposomal vitamin D3, liposomes containing C2 ceramide or liposomescontaining C6 ceramide, prepared in accordance with the proceduresdescribed in Example 1 (see above), at a lipid dose of 1.5 mg of lipidper kg of body weight of the mice, the administration being 24 hoursadministration of the p388 cells. Survival was assessed at various timespost-liposome/control administration. Results are presented in FIG. 8.

Example 8

In Vitro Cytoxicity Studies

These studies were performed using a sulforrhodamine B assay (see Monkset al, J. Natl. Cancer Inst. (U.S.) 83:757 (1987)). Compounds weredissolved in ethanol. Results from these studies, presented in thefollowing tables, are expressed as Gl₅₀ values, that is, concentrationof a drug (micromolar) required to inhibit growth of fifty percent ofthe cells.

                                      TABLE 4                                     __________________________________________________________________________    In Vitro Drug Sensitivity of Human and Mouse Cell Lines to                    Ceramide Derivatives                                                          72 Hour Drug Exposure  GI.sub.50 (uM) ± SD!                                Ceramide and     1-Acetate                                                                            1,3-Diacetate                                                                         1-Butyrate                                                                            1-Isobutyrate                         derivatives                                                                           C6-Ceramide                                                                            C6-Ceramide                                                                          C6-Ceramide                                                                           C6-Ceramide                                                                           C6-Ceramide                           __________________________________________________________________________    A549    12.0 ± 4.8                                                                          12.7 ± 7.6                                                                        6.1 ± 1.1                                                                          6.2 ± 0.4                                                                          7.9 ± 3.5                          MCF7    24.2 ± 8.5                                                                          ND     15.5 ± 0.2                                                                         17.9 ± 10.0                                                                        ND                                    MCF7/ADR                                                                              36.025 ± 0.88                                                                       ND     42.89 ± 3.6                                                                        37.80 ± 8.06                                                                       37.80 ± 4.67                       RPMI 7666                                                                             8.5 ± 3.5                                                                           9.0 ± 2.0                                                                         ND      ND      ND                                    U937    7.0 ± 1.7                                                                           11.3 ± 1.0                                                                        ND      ND      ND                                    LEWIS LUNG                                                                            9.6 ± 4.4                                                                           8.5 ± 0.4                                                                         4.9 ± 0.5                                                                          6.4 ± 2.4                                                                          ND                                    __________________________________________________________________________     ND--Not Done;                                                                 *One Experiment Only                                                     

                                      TABLE 5                                     __________________________________________________________________________    In Vitro Drug Sensitivity of Human and Mouse Cell Lines to                    Ceramide Derivatives                                                          72 Hour Drug Exposure  GI.sub.50 (uM) ± SD!                                Ceramide and   1-TBDMS                                                                              3-TBDMS                                                                              1,3-DiTBDMS                                                                           1-TBDMS-3-                               Derivatives                                                                           C6-Ceramide                                                                          C6-Ceramide                                                                          C6-Ceramide                                                                          C6-Ceramide                                                                           Butyrate C6-Cer                          __________________________________________________________________________    A549    12.0 ± 4.8                                                                        >200*  >200*  >200    >200*                                    RPMI 7666                                                                             8.5 ± 3.5                                                                         >200*  >200*  >200    >200*                                    U937    7.0 ± 1.7                                                                         >200*  >200*  134.5 ± 28.8                                                                       >200*                                    C3H10T1/2                                                                             14.0 ± 2.5                                                                          >161.2*                                                                            >200*  ND      >200*                                    LEWIS LUNG                                                                            9.6 ± 4.4                                                                         >200*  >200*  >200    >200*                                    __________________________________________________________________________     ND--Not Done;                                                                 *One Experiment Only                                                     

                                      TABLE 6                                     __________________________________________________________________________    In Vitro Drug Sensitivity of Human and Mouse Cell Lines to                    Ceramide Derivatives                                                          72 Hour Drug Exposure  GI.sub.50 (uM) ± SD!                                Ceramide and           N-Hexyl 1-Acetate-3-one                                                                        4,5-Diol C6-                          Derivatives                                                                           C6-Ceramide                                                                           Sphingosine                                                                          Sphingosine                                                                           C6-Cer   Ceramide                              __________________________________________________________________________    A549    12.0 ± 4.8                                                                         20.2 ± 0.8*                                                                       4.9 ± 0.1                                                                          14.4 ± 0.1*                                                                         25.1 ± 0.3*                        MCF7    24.2 ± 8.5                                                                         20.4 ± 0.4*                                                                       4.8 ± 0.1                                                                           6.7 ± 0.1*                                                                         20.7 ± 0.1*                        MCF7/ADR                                                                              36.03 ± 0.88                                                                       19.8 ± 0.0*                                                                       5.57 ± 1.17                                                                        14.7 ± 0.1*                                                                         28.3 ± 1.1*                        CAKI 1  6.04 ± 0.23                                                                        39.7 ± 0.8*                                                                       6.84 ± 3.62                                                                        ND       ND                                    OVCAR 3 15.15 ± 1.77                                                                       44.6 ± 0.9*                                                                        4.99 ± 0.05*                                                                      15.3 ± 0.1*                                                                         38.2 ± 2.3*                        HT 29   4.0 ± 0.2                                                                          19.3 ± 0.1*                                                                        5.2 ± 0.1*                                                                        13.9 ± 0.2*                                                                         14.6 ± 0.4*                        SKMEL 28                                                                              13.3 ± 2.51                                                                        15.6 ± 1.0*                                                                        4.94 ± 0.66*                                                                      ND       ND                                    P388    6.24 ± 0.3                                                                         ND     2.59 ± 0.3*                                                                        ND       ND                                    P388/ADR                                                                              12.7 ± 1.7*                                                                        ND     2.61 ± 1.7*                                                                        ND       ND                                    LEWIS LUNG                                                                            9.6 ± 4.4                                                                          12.2 ± 0.1                                                                        4.9 ± 0.1                                                                          14.5 ± 0.1                                                                          15.2 ± 0.1                         __________________________________________________________________________     ND--Not Done;                                                                 *One Experiment Only                                                     

                                      TABLE 7                                     __________________________________________________________________________    In Vitro Drug Sensitivity of Selected Sphingosine Derivatives on a            Diverse                                                                       Tumor Cell Line Panel                                                         72 Hour Drug Exposure                                                                                         N-C8                                                 N-C4     N-C6    N-C8    Sphingosine                                                                           N-C10                                 Cell Lines                                                                           Sphingosine                                                                            Sphingosine                                                                           Sphingosine                                                                           HCl Salt                                                                              Sphingosine                                                                            Cer-C6                       __________________________________________________________________________    A549   4.90 +/- 0.10                                                                          4.90 +/- 0.06                                                                         4.91 +/- 0.01                                                                         4.97 +/- 0.19                                                                         4.99 +/- 0.08                                                                          8.08 +/- 0.06                MCF 7  4.88 +/- 0.03                                                                          4.74 +/- 0.09                                                                         4.88 +/- 0.11                                                                         4.90 +/- 0.02                                                                         5.13 +/- 0.01                                                                          12.70 +/- 0.14               MCF 7/ADR                                                                            9.60 +/- 0.47                                                                          4.69 +/- 0.10                                                                         4.88 +/- 0.04                                                                         4.82 +/- 0.18                                                                         6.19 +/- 0.43                                                                          26.1 +/- 0.85                OVCAR 3                                                                              14.05 +/- 0.35                                                                         4.99 +/- 0.05                                                                         5.41 +/- 0.13                                                                         5.23 +/- 0.01                                                                         12.50 +/- 0.85                                                                         15.15 +/- 1.77               CAKI 1 6.88 +/- 0.52                                                                          4.28 +/- 0.04                                                                         4.64 +/- 0.07                                                                         4.47 +/- 0.01                                                                         6.18 +/- 0.01                                                                          5.88 +/- 0.10                SKMEL 28                                                                             6.13 +/- 1.03                                                                          4.94 +/- 0.06                                                                         4.96 +/- 0.03                                                                         4.95 +/- 0.02                                                                         13.00 +/- 0.00                                                                         11.55 +/- 0.07               HT 29  6.49 +/- 0.11                                                                          5.03 +/- 0.06                                                                         5.72 +/- 0.24                                                                         5.78 +/- 0.00                                                                         14.50 +/- 0.14                                                                         4.67 +/- 0.19                LEWIS LUNG                                                                           5.08 +/- 0.13                                                                          4.61 +/- 0.04                                                                         5.05 +/- 0.02                                                                         4.82 +/- 0.18                                                                         6.71 +/- 1.04                                                                          5.81 +/- 0.31                __________________________________________________________________________     GI.sub.50 (uM) +/- SD                                                    

                                      TABLE 8                                     __________________________________________________________________________    In Vitro Sensitivity of A549 and MCF 7/ADR to C6-Ceramide Derivatives at      Different Drug Exposure Times                                                 72 Hour Post-Drug Addition Incubation                                                   A549                        MCF 7/ADR                               Drug Exposure                                                                           1-Acetate                                                                            1-Butyrate                                                                           1-Isobutyrate                                                                        N-C8   1-Acetate                                                                           1-Butyrate                                                                          1-Isobutyrate                                                                       N-C8                  Time      C6-Ceramide                                                                          C6-Ceramide                                                                          C6-Ceramide                                                                          Sphingosine                                                                          C6-Ceramide                                                                         C6-Ceramide                                                                         C6-Ceramide                                                                         Sphingosine           __________________________________________________________________________     1 Hour Pulse                                                                           92.9 +/- 1.3                                                                         >100   >100   31.2 +/- 5.0                                                                         >100  >100  >100  34.3 +/- 4.2           4 Hour Pulse                                                                           44.1 +/- 1.3                                                                         52.9 +/- 2.5                                                                         >100   12.8 +/- 1.0                                                                         59.5 +/- 1.4                                                                        79.1 +/- 5.3                                                                        >100  14.2 +/- 1.2           8 Hour Pulse                                                                           35.3 +/- 2.5                                                                         36.5 +/- 0.8                                                                         40.2 +/- 2.5                                                                         6.7 +/- 0.2                                                                          42.7 +/- 1.2                                                                        44.0 +/- 1.1                                                                        52.3 +/- 1.6                                                                        8.9 +/- 0.2           24 Hour Pulse                                                                           7.2 +/- 0.2                                                                          8.6 +/- 0.6                                                                          8.4 +/- 0.7                                                                          5.0 +/- 0.1                                                                          19.5 +/- 0.8                                                                        20.8 +/- 1.8                                                                        22.7 +/- 1.1                                                                        4.8 +/- 0.1           48 Hour Pulse                                                                           4.35 +/- 0.1                                                                         4.7 +/- 0.1                                                                          5.2 +/- 0.3                                                                          4.8 +/- 0.1                                                                          15.6 +/- 0.9                                                                        17.2 +/- 0.1                                                                        18.7 +/- 0.5                                                                        4.62 +/- 0.1          72 Hour   5.1 +/- 0.1                                                                          5.5 +/- 0.2                                                                          5.6 +/- 0.2                                                                          5.0 +/- 0.3                                                                          16.7 +/- 1.7                                                                        18.5 +/- 0.4                                                                        19.6 +/- 1.0                                                                        5.0 +/- 0.1           Continuous Exposure                                                           __________________________________________________________________________     GI.sub.50 (uM) +/- SD                                                    

                  TABLE 9                                                         ______________________________________                                        Example 9                                                                     In Vivo Toxicity Studies                                                      These studies were performed by intravenous injection of n-hexyl              sphingosine, at the indicated dose (see below), into mice.                    Results are presented below.                                                  Toxicity of N-hexyl sphingosine after i.v injection in mice                   Dose           Number of Animals                                              (mg/kg)        Dead                                                           ______________________________________                                        Control (Tween-80)                                                                           0/2                                                            100            2/2                                                            50              0/2*                                                          25              0/2**                                                           12.5         0/2                                                            ______________________________________                                         *Immediate ataxia, decreased activity. Respiration: Gasping.                  After 30 minute decreased activity was still observed.                        After 24 hours no abnormality was observed.                                   **Decrease activity immediately after injection.                              After 30 minute no abnormality was observed.                             

Example 10

In Vivo Studies

These studies were performed using P-388/adriamycin resistant leukemiacells. Mice were injected i.p. with 100,000 cells, and then treated ondays 1, 3 and 5 post-injection with n-hexyl sphingosine. Results arepresented in FIG. 14.

Example 11

Compound Synthesis

Synthesis of sily ether of ceramide: The mixture of Ceramide andt-Butyldimethylsilyl chloride (1 equivalent) and imidazole (2equivalent) in DMF was stirred under N₂ at room temperature overnight.The solvent was removed under a stream of N₂ and residue was dissolvedin CH₂ Cl₂, washed (H₂ O), dried (MgSO₄) and concentrated to dryness.The residue was purified over silica gel (AcOEt: Hexan=1:3).

Synthesis of 1-ester ceramide: The mixture of ceramide and Ac2O (1equivalent) and catalytic amount of dimethyl amino pyridine in dryCH2Cl2 was stirred at room temperature for 1 hour and the reaction waschecked by TLC (AcOEt). The mixture was concentrated. The crude productwas purified over silica gel (AcOEt: Hexane=2:3.5).

Oxidation of C3-OH of ceramide to ketone: 1-OAc ceramide was dissolvedin acetone and cooled in ice-bath. Jone's reagent was dropwised slowlytill the orange color persistent. The reaction was quenched byisopropanol, and NaHCO₃ was added and stirred for 5 minutes. Thesolution was filtrate and concentrated to dryness. The crude product waspurified by preparative TLC (ACOET:Hexane=1:2.5).

Reduction of ceramide to sphingosine analogs: To an ice-cold stirredsolution of ceramide in anhydrous THF was added LiAIH₄ and the mixturewas stirred at room temperature under N₂ for 24 hours. Under ice coolingthe reaction mixtures was quenched by addition of saturated aqueousNaHCO₃. The resulting slurry was filtered and washed with THF. Thesolution was concentrated and the residue was brought into CH₂ Cl₂,washed with H₂ O, dried (MgSO₄) and concentrated to dryness. The residuewas purified over preparative TLC (silica gel) CH₂ Cl₂:MeOH:TEA=8:1:0.08.

Synthesis of 4,5-diol ceramide: To a solution of ceramide in a mixtureof Me₂ CO distilled H₂ O and t-BuOH, N-Methyl morpholine N-oxide (NMO,1.2 equivalent) and OsO₄ (catalytic amount) in THF were added. Thereaction mixture was stirred at 45° C. for 6 hours and it was quenchedby solid NaHCO₃ and the mixture was stirred for 15 minutes. Thesuspension was filtered and filtrate was dissolved in THF. The solutionwas washed with brine. The organic solution was separated, dried andconcentrated to dryness. The residue was purified over preparative TLC(THF).

What is claimed is:
 1. A pharmaceutical composition comprising acomposition having the formula R¹ --Y¹ --CHZ¹ --CH(NY² Y³)--CH₂ --Z²,wherein:R¹ is a straight-chained alkyl, alkenyl or alkynyl group havingfrom 8 to 19 carbon atoms in the aliphatic chain; Y¹ is --CH═CH--,--C.tbd.C-- or --CH(OH)CH(OH)--; Z¹ is OH or a phosphorylcholineattachment-inhibiting group selected from the group consisting of --X¹,--OX¹, X² X³ and --OX² X³ (conversion-inhibiting group); Z² is aphosphorylcholine attachment-inhibiting group selected from the groupconsisting of --X¹, --OX¹, --X² X³ and --OX² X² (conversion-inhibitinggroup); Y² is H, a phenyl group, an alkyl-substituted phenyl grouphaving from 1 to about 6 carbons in the alkyl chain, or an alkyl chainhaving from 1 to 6 carbons; Y³ is H or a group having the formula--C(O)R² or --S(O)₂ R² ; R² is a straight-chained alkyl group selectedfrom the group consisting of --(CH₂)₃ CH₁, --(CH₂)₅ CH₃, --CH₂)₇ CH₃ and--CH₂)₂ CH₂, an alkenyl chain having from 1 to 23 carbons in the chainor alkynyl chain (group) having from 1 to 23 carbon atoms in the chain;X¹ is --C(O)H, --CO₂ H, --CH₃, --C(CH₃)₃, --Si(CH₃)₃, --SiCH₃(C(CH₃)₃)₂, --Si(C(CH₃)₃)₃, --Si(PO₄)₂ C(CH₃)₃, a phenyl group, analkyl-substituted phenyl group having from 1 to 6 carbons in the alkylchain, an alkyl chain having from 1 to 6 carbons in the chain, an aminomoiety, a fluorine atom, a chlorine atom or a group having the formulaC(R³ R⁴)OH, wherein for X¹, each of R³ and R⁴ is independently an alkylchain having from 1 to 6 carbons in the chain; X² is selected from thegroup consisting of CH² --, C(CH₃)₂ --, Si(PO₄)₂ --, Si(CH₃)₂ --, SiCH₃PO₄ --, C(O)-- and S(O)₂ --; X³ is selected from the group consisting of--C(O)H, --CO₂ H, --CH₃, --C(CH₃)₃, --Si(CH₃)₃, --SiCH₃ (C(CH₃)₃)₂,--Si(C(CH₃)₃)₃, --Si(PO₄)₂ C(CH₃)₃, a phenyl group, an alkyl-substitutedphenyl group having from 1 to 6 carbons in the alkyl chain, an alkylchain having from 1 to 6 carbons in the chain, an amino moiety, achlorine atom, a fluorine atom, or a group having the formula C(R³R⁴)OH, wherein for X¹, each of R³ and R⁴ is independently an alkyl chainhaving from 1 to 6 carbons in the chain, a phenyl group or analkyl-substituted phenyl group having from 1 to 6 carbons in the alkylchain; and wherein when Z² is an amino group, R² is an aliphatic chainhaving from 1 to 9 or from 19 to 23 carbon atoms in the aliphatic chain.2. The composition of claim 1, wherein R¹ is CH₃ (CH₂)₁₂ --.
 3. Thecomposition of claim 1, wherein Y¹ is --CH═CH--.
 4. The composition ofclaim 1, wherein Y² is H.
 5. The composition of claim 1, wherein Y³ is--C(O)R² and wherein R² is an alkyl chain.
 6. The composition of claim1, wherein the conversion-inhibiting group is --OC(O)CH₃, --OC(O)CH₂ CH₂CH₃, --OC(O)CH(CH₃)CH₃, or --OSi(CH₃)₂ C(CH₃)₃.
 7. The composition ofclaim 6, wherein the conversion-inhibiting group is --OSi(CH₃)₂ C(CH₃)₃.8. The composition of claim 1, wherein the compound has the formula CH₃(CH₂)₁₂ --CH═CH--CH₂ Z¹ --CH(NHY³)--CH₂ --Z².
 9. The composition ofclaim 8, wherein Y³ is --C(O)(CH₂)₄ CH₃ and wherein Z² is OC(O)CH₃,--OC(O)CH₂ CH₂ CH₃, --OC(O)CH(CH₃)CH₃, or --OSi(CH₃)₂ C(CH₃)₃.
 10. Thecomposition of claim 1 comprising a pharmaceutically acceptable carrier.11. The composition of claim 1 comprising an additional bioactive agent.12. A method of administering a bioactive composition to an animal whichcomprises administering to the animal the composition of claim
 1. 13.The method of claim 12, wherein the animal is afflicted with a cancerand wherein the composition comprises an anticancer effective amount ofthe compound.
 14. The method of claim 13, wherein the anticancereffective amount of the composition is at least about 0.1 mg of thecomposition per kg of body weight of the animal.
 15. The method of claim14, wherein the anticancer effective amount is from about 1 mg per kg toabout 50 mg per kg.
 16. The method of claim 15, wherein the cancer is adrug resistant cancer.
 17. The method of claim 16, comprisingadministering an additional bioactive agent to the animal.